David M. Regen

STUDIES OF THE GLUCOSE-TRANSPORT SYSTEM IN THE RABBIT ERYTHROCYTE.

Abstract 1. 1. The saturation kinetics of 3- O -methylglucose transport in the rabbit erythrocyte were studied. Efflux was unaffected by external sugar and influx was unaffected by internal sugar. These findings provide new evidence that the transport site is a mobile carrier and confirm the original assumptions of Widdas that the movements of carrier and sugar-carrier complex are rate-limiting and governed by the same diffusion constant. 2. 2. The kinetics of transport couterflow induced by glucose were studied and compared to the kinetics of glucose entry. The constant governing counterflow was found to be essentially equal to the Michaelis constant for glucose entry as predicted by Widdas and by Rosenberg and Wilbrandt . The findings were, therefore, compatible with the simplifying assumptions described above. 3. 3. When cells were incubated in 42 mM 3- O -methylglucose and the internal concentration had risen to 10 mM, the ratio of influx to efflux was found to be 1.8. The discrepancy between the flux ratio and the concentration ratio is further evidence for the operation of a mobile carrier in this cell. 4. 4. The inhibitory power of 3- O -methylglucose on xylose entry was esentially as predicted from 3- O -methylglucose transport kinetics. 5. 5. A portion of the sugar movements appeared to occur by simple diffusion.

Anomalous transport kinetics and the glucose carrier hypothesis

In recent years several observations of glucose transport in human erythrocytes have been difficult to explain by the carrier model. Three new models have been described as candidates to supplant the carrier hypothesis. In this paper, some of the anomalous findings of Miller ((1968) Biophys. J. 8, 1329–1338) and of Steins group ((1972) Biochim. Biophys. Acta 266, 161–173; (1966) Biochim. Biophys. Acta 127, 179–193) are discussed in terms of the carrier model with and without unstirred layers (unstirred layers being diffusion barriers between the carrier and the bulk aqueous phases inside and outside the cell). The carrier model with no simplifying assumptions (Regen, D. M. and Morgan, H. E. (1964) Biochim. Biophys. Acta 79, 151–166) with the addition of unstirred layers suggested by Miller, accommodates virtually all the data which have been considered grounds for its rejection. All transport constants for glucose and galactose are evaluated, including the diffusion constants in the unstirred layers. Some novel ways of interpreting kinetics data are described. The asymmetric features of the carrier described by Geck are confirmed ((1971) Biochim. Biophys. Acta 241, 462–472).

Regulatory significance of transfer RNA charging levels I. Measurements of charging levels in livers of chow-fed rats, fasting rats, and rats fed balanced or imbalanced mixtures of amino acids

Abstract 1. 1. To evaluate the possible role of tRNA charging levels (percent of a tRNA class which is acylated at the acceptor site) in the control of liver anabolism, an assay for the level of charging was developed based on the ability of IO 4 -to inactivate uncharged but not charged tRNA. Charging levels of tRNA Ser and tRNA Thr could not be assayed owing to oxidation of the amino acyl moieties and attendant deacylation, and that of tRNA Cys could not be assayed owing to a high and erratic blank. The remaining charging levels were systematically somewhat underestimated by the assay. 2. 2. Evidence was obtained that charging levels in fed or fasting (24 h) rats were all near 100%. The assay was not precise enough to detect any effects of brief fasting. Tube feeding of amino acid mixtures lacking certain amino acids singly caused the charging level of the tRNA corresponding to the missing amino acid to fall considerably below that seen in the fasting rat. This provides some insight into the events leading to suppressed consumption of diets in which single amino acids are severely limiting for growth. Tryptophan deficiency produced the most striking charging level effect of those observed. The apparent high sensitivity of tRNA Trp charging level to dietary tryptophan may account in part for the peculiarly deleterious effects of tryptophan deficiency on hepatic polysome aggregation and activity.

Calculation of left ventricular wall stress.

Chamber-stress equations relate wall stresses to pressure and wall dimensions. Such equations play a central role in the analysis and understanding of heart-chamber function. Over the past three decades, several stress equations giving radically different results have been derived, used, and/or espoused. They can be classified into two categories, according to the definition of stress underlying the equation. The stresses in one class of equations are total forces per unit normal area, excluding ambient pressure but including pressure in the wall exerted by more external elements of the wall. The stresses in the other class of equations are fiber-pulling forces per unit normal area, that is, total forces per unit normal area excluding all pressure. The validity of stress equations can be tested at least three ways: 1) Do they predict that the pressure inside a small chamber nested in a larger chamber would be the sum of transmural pressures of the two chambers? 2) Do they satisfy the expectation from Laplaces law that a sphere with a given circular stress and thickness/radius ratio would exert twice the pressure of a cylinder with the same circular stress and thickness/radius ratio? 3) Do they predict that the ratio of principle stresses depends on chamber shape but not on wall/cavity ratio, with the circular/longitudinal stress ratio of a cylinder being 2 and that of a prolate spheroid being between 1 and 2? Stress equations of the first class fail all of these tests by large margins, whereas those of the second class pass all of these tests exactly. When selecting a stress equation, one should consider these distinctions as they might affect 1) evaluation of stress afterload and stress preload, 2) evaluation of contractility, 3) understanding the roles of contractility and wall/cavity ratio as determinants of pressure-making ability, 4) understanding the role of fiber orientation as a determinant of chamber shape, and 5) translations between pressure-volume relations and stress-length relations.

Characterization of β-hydroxybutyrate transport in rat erythrocytes and thymocytes

A method was developed for study of beta-hydroxybutyrate transport in erythrocytes and thymocytes. Critical to the method was a centrifugal separation of cells from medium which took advantage of beta-hydroxybutyrate transports temperature dependence and inhibition by phloretin and methylisobutylxanthine, all of which are demonstrated in this work. These properies suggested mediated transport, as did saturation kinetics and inhibition by several agents including pyruvate and alpha-cyanocinnamate. Most conclusive in this regard was a 2-fold preference for D- over L-beta-hydroxybutyrate. Entry was not Na+ dependent. It was stimulated by substitution of SO2-4 for most of the Cl-. The equilibrium beta-hydroxybutyrate space was much higher than the Cl- space of thymocytes, suggesting that beta-hydroxybutyrate entry is not associated with net inward negative current and is not coupled to outward Cl- or inward K+ movement (assuming that K+ is at elecrochemical equilibrium). Coupling to H+ entry or OH- exit is compatible with the result. These findings are consistent with beta-hydroxybutyrate entry by the carboxylate transport site which has been studied extensively with pyruvate and lactate as permeants. The Cl-/HCO-3 exchange carrier did not appear to contribute significantly to beta-hydroxybutyrate transport.

Effects of glucagon and fasting on acetate metabolism in perfused rat liver.

Abstract 1. (1) Effects of glucagon treatment in vitro and of fasting on [2- 14 C]acetate metabolism were studied in perfused rat liver. Glucagon stimulated ketogenesis about 2.8 fold and the incorporation of label into ketone bodies only 1.7 fold. The diminished specific activity of ketone bodies produced in the presence of glucagon was interpreted as evidence for increased production and disposal (turnover) of acetyl-CoA. Inhibition of acetyl-CoA utilization by other routes, therefore, was not a major cause of the increased ketogenesis. From a consideration of label incorporation into derivatives of the Krebs cycle in relation to the specific activity of acetyl-CoA (as judged from ketogenic rate and label incorporated into ketone bodies) glucagon appeared to accelerate the initial steps of the Krebs cycle about 2 fold. It diminished label incorporation into fatty acids to a degree corresponding to the reduction in acetyl-CoA specific activity. Incorporation of label into cholesterol was not affected by glucagon. 2. (2) In livers from fasting rats, ketogenesis was increased about 6.7 fold and label incorporation into ketone bodies was increased about 5.3 fold over values in fed controls. Thus, the turnover of acetyl-CoA appeard to be increased slightly by fasting. Krebs cycle activity was essentially unaffected by fasting. The inhibition of fatty acid and cholesterol synthesis in these livers appeared sufficient to contribute significantly to the ketogenic rate by sparing acetyl-CoA. 3. (3) Gluconeogenesis from endogenous substrate was markedly stimulated by glucagon and moderately stimulated by fasting. 4. (4) Secretion of newly synthesized total fatty acids, cholesterol and cholesterol ester into the circulation was markedly inhibited by glucagon.

Sugar-transport kinetics of the rat thymocyte.

Abstract Suspensions of 2 to 5% rat thymocytes were incubated at 35 °C in buffered balanced salt solution (pH 7.3) with lactate and β-hydroxybutyrate as fuels. The dependence of 3- O -[ Me - 3 H]methylglucose influx on external and internal 3- O -methylglucose concentrations was studied. Entry was almost rectilinear during the first minute. From the dependence of methylglucose entry (into sugar-free cells) on external methylglucose concentration, we judged the entry K m to be about 7.7 m m and the entry V to be about 0.64 μmol · min −1 · (ml of packed cell volume) −1 . Methylglucose inside the cell enhanced influx, hence equilibrium exchange was faster than entry. The dependence of equilibrium exchange on methylglucose concentration (inside and outside being equal) indicated a K m of about 25 m m and a V of about 2.1 μmol · (min) −1 · (ml of cell volume) −1 . This effect of internal sugar indicated that entry into sugar-free cells is limited mainly by the return of empty carrier to the outside surface and that loading the carrier on the inside enhances its outward mobility. The K m and V for influx into cells containing 21 m m methylglucose were 5.9 m m and 1.17 μmol · min −1 · (ml of packed cells) −1 . The effect of 21 m m internal sugar on lowering the influx K m from about 7.7 m m to about 6 m m was reproducible and contributed to the evaluation of the constants of the transport rate law. It indicated that loading of the carrier at the external surface reduces its mobility, in contrast to the effect of loading on the inside. Mechanical explanations for this behavior are discussed.

Effects of pH on β-hydroxybutyrate transport in rat erythrocytes and thymocytes

Entry of beta-hydroxybutyrate into erythrocytes and thymocytes is facilitated by a carrier (C), as judged from temperature dependence, saturation kinetics, stereospecificity, competition with lactate and pyruvate, and inhibition by moderate concentrations of methylisobutylxanthine, phloretin, or alpha-cyanocinnamate. We studied the dependence of influx and efflux on internal and external pH and [beta-hydroxybutyrate]. Lowering external pH from 8.0 to 7.3 to 6.6 enhanced influx into erythrocytes by lowering entry Km from 29 to 16 to 10 mM, entry V being independent of external pH. Lowering external pH inhibited efflux. At low external pH, external beta-hydroxybutyrate enhanced efflux slightly. At high external pH, external beta-hydroxybutyrate inhibited efflux. Internal acidification inhibited influx and internal alkalization enhanced influx. Internal beta-hydroxybutyrate (betaHB) enhanced influx more in acidified than alkalized cells. These data are compatible with coupled betaHB-/OH- exchange, betaHB- and OH- competing for influx, C:OH- moving faster than C: betaHB-, empty C being immobile. They are also compatible with coupled betaHB-/H+ copermeation, empty C moving inward faster than H+:C:betaHB-, H+:C being immobile, and C:betaHB- (without H+) being so unstable as not to be formed in significant amounts (relative to C, H+:C, and H+:C:betaHB-).

Sugar transport in beef erythrocytes

1. 1. The entry of sugar into beef erythrocytes was found to be facilitated by a saturable, passive process with unusually high affinity for d-glucose and d-3-O-methylglucose, the Michaelis constants for these two sugars being 0.09 and 0.17 mM respectively. In other mammalian cells, the transport Km is about the same for these two sugars, and ranges between 6 and 15 mM. The transport V was about 0.1–0.2 μmole/min per ml for both sugars, slow by comparison to most other mammalian cells. 2. 2. The beef erythrocyte differed from the human cell but resembled the rabbit cell in that extracellular methyl glucose did not affect efflux, and intracellular methylglucose appeared not to affect influx. However, the inhibitory effects of glucose on methylglucose entry were stronger if the glucose was added some time prior to the addition of methylglucose. Since intracellular methyl glucose did not inhibit influx, the time dependent inhibition by glucose was likely due to glucose metabolism rather than intracellular glucose per se. Other kinetic criteria affirmed this conclusion.

Characteristics of single isovolumic left-ventricular pressure waves of dog hearts in situ

SummaryBy fitting isovolumic phases of an ejecting beat with a model-wave function, one can predict source pressure of the ejecting beat (Sunagawa et al.Trans Biomed Eng 1980; 27:299–305), this being a major determinant of systolic performance. Prior applications of this principle have involved two assumptions: (1) that the isovolumic pressure wave is shaped like an inverted cosine wave, and (2) that duration of an isovolumic beat is the same as that of an ejecting beat. The first assumption might cause overestimation of source pressure, since an isovolumic pressure wave begins declining before the midpoint of the wave. The second assumption might cause underestimation of source pressure, since an ejecting beat is always shorter than an adjacent isovolumic beat at the ejecting beats enddiastolic volume. Although the two errors tend to cancel, it would be more rational and accurate to use a realistic model wave shape and a realistic isovolumic beat duration. To acquire the information necessary for this, pressure and volume time courses were measured during ejecting beats and adjacent isovolumic beats in dogs under the following steady-state conditions: basal, atrial pacing at various rates, infusion of dobutamine, infusion of verapamil, coronary ligation(s), and ventricular pacing at various sites. These conditions affected the amplitude and duration of isovolumic pressure waves substantially but did not affect the shape of the waves significantly. The duration of each isovolumic beat exceeded that of the previous ejecting beat to a degree which corresponded approximately to the ejecting beats normalized pressure reserve (source pressure minus peak ejection pressure)/(source pressure). A more accurate source-pressure prediction should be possible by use of a realistic isovolumic pressure-wave shape and by taking account of the effect of pressure reserve on contraction duration.